An Adenovirus Vector with Genetically Modified Fibers Demonstrates Expanded Tropism via Utilization of a Coxsackievirus and Adenovirus Receptor-Independent Cell Entry Mechanism

Abstract

Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor.

Full text

JOURNAL OF VIROLOGY,
0022-538X/98/$04.0010
Dec. 1998, p. 9706–9713 Vol. 72, No. 12
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
An Adenovirus Vector with Genetically Modified Fibers Demonstrates
Expanded Tropism via Utilization of a Coxsackievirus
and Adenovirus Receptor-Independent
Cell Entry Mechanism
IGOR DMITRIEV, VICTOR KRASNYKH, C. RYAN MILLER, MINGHUI WANG,
ELENA KASHENTSEVA, GALINA MIKHEEVA, NATALYA BELOUSOVA,
AND DAVID T. CURIEL*
Gene Therapy Program, Comprehensive Cancer Center, University of Alabama at Birmingham,
Birmingham, Alabama 35294-3300
Received 11 June 1998/Accepted 2 September 1998
Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy
applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer
to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve
CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of
viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an
Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the
virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its
expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An
increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector,
suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for
treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor.
Adenovirus (Ad) vectors are useful in a wide variety of gene
therapy applications. One of the principal attributes recom-
mending the employment of these vectors is their unparalleled
efficacy in accomplishing gene transfer in vivo. This property
has been noted in a variety of different organs.
There are, however, some limitations associated with the use
of recombinant Ad for gene therapy. One such disadvantage is
related to the reliance of the virus on the presence of the
coxsackievirus and adenovirus receptor (CAR) to achieve high
levels of gene transfer. In certain settings, this may result in
sequestration of recombinant virions by nontarget, yet high-
CAR-expressing cells, whereas the true target cells, if low in
CAR, are poorly transduced. In order to compensate for this
sequestration, significant escalation in the dose of adminis-
tered vector is needed, which increases the risk of inducing
both direct toxicity and immune responses against the vector,
thus further compromising the overall efficacy of the therapy.
Therefore, the utility of the present generation of Ad vec-
tors for gene therapy may be significantly improved by
achieving targeted transduction of specific cell types by the
virus.
In this regard, the initial steps of Ad infection involve at least
two sequential virus-cell interactions, each being mediated by a
specific protein component of Ad capsid. The primary binding
of the virus to the cell surface receptor, CAR (9, 10, 38), is
mediated by the knob domain of the fiber protein (23). This is
followed by the internalization of the virus within a clathrin-
coated endosome (39). The virus then escapes from the endo-
some by triggering its acidification via a secondary interaction
of the argininine-glycine-aspartic acid (RGD) motif of Ad pen-
ton base protein with cellular integrins a
v
b
3
and a
v
b
5
(4, 5, 41,
42). Following the endosome escape, partially dismantled virus
translocates to the nuclear pore complex and releases its ge-
nome into the nucleoplasm where subsequent steps of viral
replication take place.
As the fiber and the penton base are key mediators of the
cell entry mechanism developed by Ad, targeting of recombi-
nant Ad vectors may be achieved via genetic modifications of
these capsid proteins. In order to overcome the limitations
imposed by the CAR dependence of Ad infection, Michael et
al. (27) proposed the incorporation of small peptide motifs
possessing receptor binding specificities into the carboxy ter-
minus of Ad fiber protein, thus enabling the virus to attach and
infect via a novel cell surface receptor. This concept has been
further developed by Wickham et al. (43, 44), who have proved
the feasibility of this approach by generating several recombi-
nant Ad containing fibers with targeting ligands positioned at
the carboxy terminus of the fiber molecule.
Although in some cases genetic modification of the carboxy
terminus of Ad fiber has proved its utility with respect to vector
retargeting, it failed in others (44), thereby suggesting that this
locale in the fiber molecule is not the optimal site for incor-
poration of targeting protein moieties. In this regard, pub-
lished findings (15, 44) strongly suggest that the addition of
more than 25 to 30 amino acid residues of heterologous pro-
tein sequence to the carboxy terminus of the fiber molecule has
dramatic negative effect on the stability of the fiber trimer and,
therefore, is incompatible with the fiber functions. In addition,
the three-dimensional structure of the fiber knob (45) clearly
indicates that the carboxy terminus of the fiber points toward
the virion, that is, away from the cell surface, thereby providing
a suboptimal environment for the incorporation of targeting
ligands.
With these findings in mind, we recently reported that an-
other locale within the fiber molecule, the HI loop of the fiber
* Corresponding author. Mailing address: Gene Therapy Program,
Comprehensive Cancer Center, University of Alabama at Birming-
ham, Birmingham, AL 35294-3300. Phone: (205) 934-8627. Fax: (205)
975-7476. E-mail: david.curiel@ccc.uab.edu.
9706

knob domain, could be used as a convenient site for incorpo-
ration of heterologous ligands (21). As the next logical step, we
explored the utility of the HI loop for incorporation of target-
ing ligands to allow modification of Ad tropism. Specifically,
we sought to capitalize on the recently published reports on
phage biopanning (3, 31) by choosing an RGD motif proven to
have in vivo targeting capabilities. We have shown that incor-
poration of this peptide into the fiber knob allowed the virus to
utilize the RGD-integrin interactions as an alternative infec-
tion pathway, thereby dramatically improving the ability of the
virus to transduce several types of cells, which normally are
refractory to Ad infection. In order to show the utility of the
modified virion for application which may have immediate
clinical translation, we employed this viral vector as a means
for efficient gene transfer to primary ovarian cancer cells. Spe-
cifically, we have shown that recombinant Ad vector containing
fibers with RGD motif in the HI loop is capable of dramatically
augmenting gene delivery to target cells via a CAR-indepen-
dent cell entry mechanism.
MATERIALS AND METHODS
Cells and tissues. The 293 human kidney cell line transformed with Ad type 5
(Ad5) DNA was purchased from Microbix (Toronto, Ontario, Canada). Human
ovarian carcinoma cell lines SKOV3.ip1 and OV-4 were obtained from Janet
Price (M. D. Anderson Cancer Center, Houston, Tex.) and Timothy J. Eberlein
(Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.),
respectively. Human umbilical vein endothelial cells (HUVEC) and human em-
bryonal rhabdomyosarcoma (RD) cells were from American Type Culture Col-
lection (Rockville, Md.). All cell lines were grown at 37°C in media recom-
mended by the suppliers in a humidified atmosphere of 5% CO
2
.
Ascitic fluid samples from patients with epithelial ovarian carcinoma were
obtained at the Hospital of the University of Alabama at Birmingham (UAB),
Division of Gynecologic Oncology. All samples were classified by pathologists at
UAB Hospital, Department of Pathology. The samples were processed imme-
diately once received or stored at 270°C until needed.
Enzymes. Restriction endonucleases, Klenow enzyme, T4 DNA ligase, T4
polynucleotide kinase, and proteinase K were from either New England Biolabs
(Beverly, Mass.) or Boehringer Mannheim (Indianapolis, Ind.).
Monoclonal antibodies. Anti-a
v
b
3
integrin monoclonal antibody LM609 and
anti-a
v
b
5
integrin monoclonal antibody P1F6 were purchased from Chemicon
International, Inc. (Temecula, Calif.) and Gibco-BRL (Gaithersburg, Md.), re-
spectively. Anti-CAR monoclonal antibody RmcB (9) was obtained from R. W.
Finberg (Dana-Farber Cancer Institute, Harvard Medical School, Boston,
Mass.).
Viruses. A recombinant Ad5 vector, AdCMVLuc, containing a firefly lucif-
erase-expressing cassette in place of the E1 region of the Ad genome, was
obtained from R. D. Gerard (University of Texas Southwestern Medical Center,
Dallas). Ad vector, Ad5lucRGD, containing recombinant fiber-RGD protein
and expressing the firefly luciferase was generated by transfection of 293 cells
with PacI-digested pVK703 by the method described previously (12). Ad were
propagated on 293 cells and purified by centrifugation in CsCl gradients by a
standard protocol. Determination of virus particle titer was accomplished spec-
trophotometrically by the method of Maizel et al. (24), using a conversion factor
of 1.1 3 10
12
viral particles per absorbance unit at 260 nm. To determine the titer
of infectious viral particles, the plaque assay on 293 cells was performed by the
method of Mittereder et al. (28) was used.
Construction of recombinant plasmids. In order to generate a recombinant
Ad5 fiber gene encoding the fiber with the RGD-4C peptide within the HI loop
of the knob domain, a duplex made of oligonucleotides CAC ACT AAA CGG
TAC ACA GGA AAC AGG AGA CAC AAC TTG TGA CTG CCG CGG
AGA CTG TTT CTG CCC and GGG CAG AAA CAG TCT CCG CGG CAG
TCA CAA GTT GTG TCT CCT GTT TCC TGT GTA CCG TTT AGT GTG
was cloned into the EcoRV site of previously designed plasmid pQE.KNOBDHI
(21), thereby generating pQE.KNOB.RGD
HI
.
To make a shuttle vector suitable for the generation of the viral genome of
interest, a BstXI-MunI fragment of the modified fiber gene containing RGD-4C
coding sequence was subcloned from pQE.KNOB.RGD
HI
into the fiber shuttle
vector pNEB.PK3.6 (22) cleaved with BstXI and MunI. In order to obtain Ad5
genome containing the fiber-RGD gene, the resultant plasmid, pNEB.PK.
F
HI
RGD, was then utilized for homologous DNA recombination with SwaI-
digested pVK50 (21) in Escherichia coli BJ5183 as previously described (12). The
plasmid obtained as a result of this recombination was designated pVK503.
Firefly luciferase gene was excised from plasmid pGEM
R
-luc (Promega, Mad-
ison, Wis.) as a 1.7-kb BamHI-XhoI fragment and cloned into BamHI-XhoI-
digested pcDNA3 (Invitrogen, Carlsbad, Calif.), resulting in pcDNA.Luc. To
destroy PacI and ClaI sites in the luciferase open reading frame, a synthetic
duplex consisting of oligonucleotides CAA ATA CAA AGG ATA TCA GGT
GGC CCC CGC TGA ATT GGA GT and CGA CTC CAA TTC AGC GGG
GGC CAC CTG ATA TCC TTT GTA TTT GAT was used to replace the 41-bp
PacI-ClaI fragment in pcDNA.Luc, thereby generating pcLucPC1.
In order to make a shuttle vector containing this modified luciferase gene in
the context of expression cassette, the gene was cloned in pACCMVpLpA (8) as
follows. Plasmid pcLucPC1 was cleaved with BamHI, treated with Klenow en-
zyme to fill in the ends, and then cut with XhoI. The cloning vector, pACCM-
VpLpA, was cut with EcoRI, treated with Klenow enzyme, and then cleaved with
SalI. The ligation of these two DNA molecules resulted in pACCMV.LucDPC.
This plasmid was then used for homologous DNA recombination with ClaI-
linearized pVK503 in order to generate pVK703, containing the genome of
Ad5lucRGD.
To derive a recombinant baculovirus expressing fiber-RGD, previously made
transfer vector pFB.F5
HI
FLAG (21) was modified in the following way. First,
EcoRI linker, CGG CGA ATT CGC, was incorporated into the ClaI site of
pFB.F5
HI
FLAG, resulting in pFB.F5.RI. Then, the NcoI-MunI fragment of
pNEB.PK.F
HI
RGD containing the 39 portion of the fiber-RGD gene was used to
replace an NcoI-MunI fragment in pFB.F5.RI, generating pFB.F5
HI
RGD. This
plasmid was then used to generate recombinant baculovirus genome via site-
specific transposition by utilizing a Bac-to-Bac kit (Gibco-BRL) according to the
manufacturer’s recommendations.
Flow cytometry. Cells grown in T75 flasks were released from the flasks by the
addition of EDTA and resuspended in SM buffer (HEPES-buffered saline, 0.1%
sodium azide, 1% bovine serum albumin [BSA]) at 2 3 10
6
cells/ml. Two
hundred thousand cells were incubated with 5 mg of LM609, P1F6, RmcB, or no
primary monoclonal antibody (negative control) per ml in 200 mlofSMfor2h
at 4°C. Cells were then washed with SM and incubated with secondary fluores-
cein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G serum
(Jackson Labs, West Grove, Pa.) (5 mg/ml) for1hat4°C. After the cells were
washed with SM, 10
4
cells per sample were analyzed by flow cytometry at the
UAB FACS Core Facility.
Recombinant proteins. Recombinant Ad5 fiber knob protein was expressed in
E. coli and purified by immobilized metal ion affinity chromatography (IMAC)
on Ni-nitrilotriacetic acid (NTA)–Sepharose (Qiagen, Valencia, Calif.) as rec-
ommended by the manufacturer.
Human Ad2 penton base protein was expressed in Spodoptera frugiperda Sf9
cells by recombinant baculovirus AcNPV-PbWT (18) provided by P. Boulanger
(Institute of Biology, Montpellier, France). The penton base protein was purified
from baculovirus-infected cells by two-step ion-exchange chromatography utiliz-
ing a DEAE-Sepharose FF column (Pharmacia, Piscataway, N.J.) followed by
purification on POROS HQ column (PerSeptive Biosystems, Framingham,
Mass.).
Recombinant fiber proteins expressed in baculovirus-infected Sf9 cells were
purified by chromatography on Ni-NTA-Sepharose as previously described (21).
The protein concentrations were determined by the Bradford protein assay
(Bio-Rad, Hercules, Calif.) with bovine gamma globulin as the standard.
ELISA. Solid-phase binding assay was performed by a method previously
described by Sharma et al. (36). Briefly, purified fiber proteins or Ad virions were
diluted in 50 mM carbonate-bicarbonate buffer, pH 9.6, to a concentration of 10
mg of protein per ml, and 100-ml aliquots were added to the wells of a 96-well
Nunc-Maxisorp enzyme-linked immunosorbent assay (ELISA) plate. Plates were
incubated overnight at 4°C and then blocked for2hatroom temperature by the
addition of 200 ml of blocking buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl,
0.5% casein) to each well. Wells were then washed three times with the washing
buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl). Purified integrin a
v
b
3
(Chemi-
con) diluted in binding buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM
CaCl
2
, 1 mM MgCl
2
, 1 mM MnCl
2
, 0.5% casein) to concentrations ranging from
0.04 to 0.5 mg/ml was added to the wells in 100-ml aliquots. After overnight
incubation at 4°C, the wells were washed three times with washing buffer con-
taining 2 mM CaCl
2
, 1 mM MgCl
2
, and 1 mM MnCl
2
. Bound integrin was
detected with mouse monoclonal anti-human integrin a
v
subunit antibody
VNR139 (Gibco-BRL). VNR139 antibody diluted 1:3,000 in binding buffer was
added to the wells in 100-ml aliquots, incubation was continued for1hatroom
temperature, and then the wells were washed again. The ELISA plate was then
developed with VECTASTAIN kit (Vector Laboratories, Burlingame, Calif.) as
recommended by the manufacturer. Color development was stopped by the
addition of1NH
2
SO
4
, and plates were read in a microtiter plate reader set at
490 nm.
Ad-mediated gene transfer assay. Ad-mediated transduction experiments uti-
lizing cell lines were performed as described previously (22).
Primary cells from ascitic fluid samples obtained from ovarian cancer patients
were prepared for this analysis as follows. First, the erythrocytes present in the
samples were lysed by the addition of buffer containing 150 mM NH
4
Cl,1mM
KHCO
3
, and 0.1 mM Na
2
EDTA. Then, the cell debris and dead cells were
separated from the live cells by slow-speed centrifugation on a step gradient of
Ficoll-Hypaque (Media Preparation Shared Facility, UAB Comprehensive Can-
cer Center, Birmingham, Ala.). The cells were washed twice with Dulbecco’s
modified Eagle’s medium/F12 (DMEM/F12) (Cellgro, Herndon, Va.) containing
10% fetal bovine serum (Hyclone Laboratories, Logan, Utah), 100 U of peni-
cillin per ml, and 100 mg of streptomycin per ml.
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Ad binding assay. Binding of
125
I-labeled Ad to 293, HUVEC, or RD cells was
assayed in a procedure described previously (21).
RESULTS
Fiber-RGD protein efficiently interacts with integrins via the
RGD tripeptide. Recently we demonstrated that the FLAG
octapeptide incorporated in the HI loop of Ad5 fiber does not
interfere with correct folding of the cell-binding site localized
in the knob and is available for binding to FLAG-specific
antibody in immunoprecipitation assay (21). To utilize these
findings for the purposes of Ad retargeting, we chose to intro-
duce in the HI loop of the fiber knob an RGD-4C peptide,
CDCGRDCFC, which is known to bind with high affinities to
several types of integrins present on the surfaces of mamma-
lian cells. This effort was undertaken in an attempt to generate
an Ad vector, which would be able to bind to cells by utilizing
fiber-RGD–integrin interaction. Therefore, the infection by
such virus would not be dependent on the presence of CAR on
a cell membrane.
We first chose to express an RGD-4C-containing fiber pro-
tein, fiber-RGD, in a baculovirus expression system in order to
characterize the protein with respect to its ability to perform
the targeting functions. The sequence encoding the amino-
terminal six-His tag was incorporated in the fiber-RGD gene in
order to facilitate downstream purification of the product. As
anticipated from our previous experiments done with fiber-
FLAG protein, electrophoresis of IMAC-purified fiber-RGD
protein showed that the fiber retains its native trimeric struc-
ture (data not shown), which is known to be crucial for asso-
ciation of the fiber with the penton base during virion assem-
bly. In order to assess the ability of the fiber-RGD protein to
bind to integrins, we employed this fiber protein for an ELISA
utilizing purified integrin a
v
b
3
. This assay showed that, in con-
trast to the wild-type fiber protein used as a negative control,
the fiber-RGD protein binds a
v
b
3
integrin very efficiently (Fig.
1). Therefore, these experiments confirmed the functional util-
ity of the modified fiber and provided a rationale for genera-
tion of recombinant Ad containing such fibers.
The virus was derived by the method described by Chartier
et al. (12). To simplify the downstream gene transfer assays, an
expression cassette containing the firefly luciferase gene driven
by cytomegalovirus promoter was introduced in place of the E1
region of the Ad genome. The genome of the new virus des-
ignated Ad5lucRGD was generated in E. coli via a two-step
protocol utilizing homologous DNA recombination between
the previously designed plasmid pVK50 (21) and fragments of
DNA isolated from two shuttle vectors, pNEB.PK.F
HI
RGD
and pACCMV.LucDPC, which contain the fiber gene and the
luciferase expression cassette flanked by Ad DNA sequences,
respectively. Utilization of this method requires the digestion
of the resultant recombinant plasmid containing the newly
generated Ad genome with restriction endonuclease PacIto
release inverted terminal repeats of Ad5 DNA from the plas-
mid backbone. In order to be able to use the firefly luciferase
gene, which contains an internal PacI site, in the context of this
method, we eliminated this site by introducing a silent muta-
tion into the gene. The plasmid obtained as a result of afore-
mentioned DNA recombinations, pVK703, was then utilized
for transfection of 293 cells to rescue Ad5lucRGD. The iden-
tity of the virus was confirmed by PCR as well as by cycle
sequencing of viral DNA isolated from CsCl-purified virions of
Ad5lucRGD.
To demonstrate the accessibility of the RGD tripeptide in-
corporated in the fiber of Ad5lucRGD, we utilized this virus in
an ELISA analogous to the one used previously for purified
fiber protein. This analysis clearly showed efficient binding of
the a
v
b
3
integrin to immobilized particles of Ad5lucRGD, while
binding of a
v
b
3
to a control virus was at the background level
at all concentrations of integrin used (Fig. 2). Based on these
results, we hypothesized that Ad5lucRGD is able to interact in
vitro and in vivo with various types of RGD-binding integrins,
thereby utilizing this interaction at early steps of infection in
order to attach to target cells.
Ad5lucRGD is capable of mediating a CAR-independent
gene delivery. Our next goal was to examine whether introduc-
tion of the RGD motif in the fiber of Ad5lucRGD resulted in
any changes with respect to the ability of this virus to infect
cells. In order to investigate the infection pathway utilized by
Ad5lucRGD, we sought to employ this virus for gene transfer
to several cell lines, expressing various levels of CAR as well as
integrins a
v
b
3
and a
v
b
5
. To achieve this goal, a panel of the cell
lines, including the 293 human kidney cells, human umbilical
cord endothelial cells, HUVECs, and human embryonal RD
FIG. 1. Analysis of interaction between recombinant fiber proteins and a
v
b
3
integrin. Baculovirus-expressed fiber proteins absorbed on an ELISA plate were
incubated with various concentrations of purified integrin a
v
b
3
. Integrin bound
to fiber proteins was then detected with anti-a subunit monoclonal antibody
VNR139. Each point represents a mean of three readings obtained in one
experiment. Some error bars depicting standard deviations are smaller than the
symbols. wt, wild type.
FIG. 2. ELISA of a
v
b
3
integrin binding to immobilized AdCMVLuc and
Ad5lucRGD virions. CsCl-purified virions of AdCMVLuc and Ad5lucRGD im-
mobilized in the wells of an ELISA plate were incubated with affinity-purified
a
v
b
3
integrin, followed by incubation with monoclonal antibody VNR139. Data
shown are means 6 standard deviations from an experiment performed in
triplicate.
9708 DMITRIEV ET AL. J. VIROL.

cells, was employed for a series of flow cytometry assays. While
293 cells readily support Ad infection, HUVECs have been
shown to bind Ad poorly (43), whereas CAR expression in RD
cells was reported to be passage dependent (35). Our flow
cytometry assay showed that 293 cells express high levels of
CAR (Fig. 3A) and a
v
b
5
integrin, while expression of a
v
b
3
is
moderate (Fig. 3B). HUVECs demonstrated moderate levels
of CAR expression (Fig. 3C), whereas both integrins were
present at the cell surface in rather large amounts (Fig. 3D).
RD cells were CAR negative (Fig. 3E), while being high a
v
b
5
and moderate a
v
b
3
expressors (Fig. 3F). Therefore, for our
subsequent gene transfer experiments, we established a set of
cell lines covering a full range of CAR expression profiles and
with moderate to high levels of integrins a
v
b
3
and a
v
b
5
present
on their cytoplasmic membranes.
Ad5lucRGD was then utilized for an assay based on com-
petitive inhibition of Ad-mediated gene delivery by recombi-
nant Ad5 fiber knob protein, known to efficiently block virus
binding to CAR.
As shown in Fig. 4A, luciferase expression in 293 cells me-
diated by our control virus, AdCMVLuc, was efficiently
blocked by recombinant knob protein. Depending on the mul-
tiplicity of infection (MOI) used, knob protein blocked 85 to
93% of luciferase activity in AdCMVLuc-transduced cells.
In marked contrast, the same concentration of knob was
able to block only 40 to 60% of Ad5lucRGD-mediated gene
expression in 293 cells, thereby indicating that in addition to
well-characterized fiber-CAR interaction utilized by the wild-
type Ad5, Ad5lucRGD is capable of using an alternative,
CAR-independent cell entry pathway. The contribution of that
alternative mechanism of cell binding was quite significant,
providing 40 to 60% of overall gene transfer to 293 cells.
To further investigate the phenomenon of Ad5lucRGD-di-
rected gene delivery, we utilized the same strategy to look into
transduction of HUVECs. It has been shown that these cells
are relatively difficult to transduce with Ad vectors containing
wild-type fibers (43, 44). These findings were corroborated
with our flow cytometry data, which showed modest levels of
CAR expression in HUVECs. Importantly, rather high levels
of a
v
b
3
and a
v
b
5
integrins detected in these cells suggested
that HUVECs should be readily transduced with Ad5lucRGD.
Although the levels of luciferase activity in HUVECs mediated
by either virus were considerably lower than those in 293 cells,
our experiment revealed striking differences between the trans-
duction profiles demonstrated by these two viruses (Fig. 4B).
First, luciferase expression in the Ad5lucRGD-transduced
FIG. 3. Flow cytometric analysis of CAR and integrin expression in 293,
HUVEC, and RD cells. Cells were incubated with anti-CAR (RmcB), anti-a
v
b
3
(LM609), or anti-a
v
b
5
(P1F6) integrin monoclonal antibodies, washed with SM
to remove unbound monoclonal antibodies, and incubated with secondary FITC-
labeled goat anti-mouse immunoglobulin G serum as described in Materials and
Methods. After removal of the FITC-labeled antibodies, aliquots of 10
4
cells
were analyzed by flow cytometry. Expression of CAR in 293 (A), HUVEC (C),
and RD (E) cells and of a
v
b
3
(thin line) and a
v
b
5
(heavy line) integrins in 293
(B), HUVEC (D), and RD (F) cells is shown. The dotted line shows the results
for the negative control.
FIG. 4. Ad-mediated gene transfer to various human cell lines. 293 (A),
HUVEC (B), or RD (C) cells preincubated for 10 min at room temperature in
either DMEM/F12 or DMEM/F12 containing recombinant Ad5 fiber knob at
100 mg/ml were then exposed for 30 min at room temperature to AdCMVLuc or
Ad5lucRGD in DMEM/F12 at 1, 10, or 100 PFU/cell. The unbound virus was
aspirated, and complete medium was added. After incubation at 37°C for 30 h,
the cells were lysed and the luciferase activity (in relative light units) was deter-
mined. Background luciferase activities detected in mock-infected cells were 261,
223, and 163 relative light units for 293, HUVEC, and RD cells, respectively.
These activities were subtracted from all readings obtained with the correspond-
ing cell line. Each point represents the mean 6 standard deviation of three
determinations.
VOL. 72, 1998 GENETICALLY MODIFIED Ad5 VECTOR WITH EXPANDED TROPISM 9709

cells was about 30-fold higher than in the cells transduced
with AdCMVLuc. Second, the effect of the Ad5 fiber knob
on AdCMVLuc-mediated transduction was less dramatic than
in our experiments with 293 cells, consistent with a relative lack
of CAR in the HUVECs. Most importantly, recombinant knob
protein had no inhibition effect on the levels of luciferase
expression directed by Ad5lucRGD.
Very similar results were then generated on RD cells, which
do not express CAR. The luciferase activity detected in the
lysates of AdCMVLuc-transduced RD cells was extremely low:
at an MOI of 1 PFU/cell, it was almost equal to background
readings obtained in mock-infected cells (Fig. 4C). Once again,
Ad5lucRGD was capable of directing the levels of transgene
expression 16- to 47-fold higher than those mediated by
AdCMVLuc. This expression was not responsive to inhibition
by the fiber knob.
These experiments clearly showed that incorporation of the
RGD-4C peptide into the fiber of Ad5lucRGD resulted in
dramatic changes in the initial steps of virus-cell interaction,
presumably by creating an alternative cell attachment pathway.
Ad5lucRGD demonstrates increased efficiencies of cell bind-
ing due to utilization of RGD-integrin interaction. Having
established that AdCMVLuc and Ad5lucRGD demonstrate
different efficiencies of gene delivery as well as different pro-
files of fiber knob-mediated inhibition of transduction, our
next task was to compare the cell binding profiles of these two
viruses. To address this issue, we labeled both viruses with
125
I
and employed them in the virus binding assay on 293,
HUVEC, and RD cells. This assay was performed under con-
ditions (4°C) allowing the viruses to bind the cells, but pre-
venting virus internalization.
As shown in Fig. 5, binding efficiencies demonstrated by
Ad5lucRGD and AdCMVLuc on CAR-positive 293 cells were
similar, while the percentages of labeled Ad5lucRGD virions
bound to HUVEC and RD cells were significantly higher than
those of AdCMVLuc virions.
Since the ultimate goal of incorporating the RGD-contain-
ing peptide within the fiber molecule was to allow the virus to
utilize cellular integrins as alternative receptors, we conducted
an assay in which binding of radiolabeled viruses to the cells
was accomplished in the presence of recombinant Ad2 penton
base protein. Due to the presence of RGD motif in the highly
mobile loop protrusion identified within its molecule (37), the
penton base is able to bind a
v
b
3
and a
v
b
5
integrins and there-
fore competes for binding to these cellular receptors with other
molecules or macromolecular complexes containing an RGD
motif.
When binding of the viruses to 293 cells was assayed (Fig.
6A), the penton base protein failed to inhibit cell binding of
either virus, whereas the fiber knob protein, alone as well as
together with the penton base, blocked 94% of AdCMVLuc
and 75% of Ad5lucRGD binding.
The same experiment performed with HUVECs showed that,
once again, the knob protein inhibited binding of AdCMVLuc
particles to a greater extent than that of Ad5lucRGD virions
(Fig. 6B). In addition, penton base was capable of decreasing
Ad5lucRGD-associated radioactivity bound to these cells by
25%, while its effect on AdCMVLuc binding was marginal.
When used together, both blocking agents caused 40% de-
crease in Ad5lucRGD binding. Similar results were obtained
when these viruses were employed for binding assay on RD
cells (data not shown). Although the penton base did not block
binding of Ad5lucRGD to HUVECs as efficiently as the knob
protein blocked binding of our control virus, its utilization as
an integrin-specific inhibitor showed that Ad5lucRGD is capa-
ble of using cellular integrins as alternative receptors during
the infection process.
Ad5lucRGD mediates enhanced gene transfer to human
ovarian cancer cells. Since a number of clinical trials utilizing
Ad vectors to treat cancer patients via direct in vivo gene
delivery are currently under way, we chose to investigate
whether the expanded tropism of Ad5lucRGD would render it
useful for this type of clinical application.
FIG. 5. Comparison of binding of
125
I-labeled adenoviruses to 293, HUVEC,
or RD cells. One-hundred-microliter aliquots of cells in DMEM-Ad medium
(DMEM, 20 mM HEPES, 0.5% BSA), 10
6
cells per aliquot, were incubated for
1 h at 4°C with 50 mlof
125
I-labeled Ad (10
5
cpm per sample). The samples were
then diluted with 4 ml of phosphate-buffered saline containing 0.1% BSA, and
the cells were pelleted by centrifugation. Radioactivities of cell pellets were
determined with a gamma counter. Data shown are means 6 standard deviations
from an experiment performed in triplicate.
FIG. 6. Inhibition of binding of labeled AdCMVLuc and Ad5lucRGD to 293
and HUVEC cells. 293 (A) or HUVEC (B) cells were preincubated with
DMEM-Ad or DMEM-Ad containing either Ad5 fiber knob (100 mg/ml) or Ad2
penton base (100 mg/ml) or both for1hat4°C. Fifty-microliter aliquots of
125
I-labeled viruses were then added to the samples. The rest of the procedure
was as described in the legend for Fig. 5.
9710 DMITRIEV ET AL. J. VIROL.

First, we looked into the ability of this recombinant vector to
deliver genes to cultured human ovarian cancer cells. Charac-
terization of two cell lines, SKOV3.ip1 and OV-4, by flow
cytometry showed that they both express moderate to high
levels of integrins a
v
b
3
and a
v
b
5
(Fig. 7B and D), SKOV3.ip1
expresses a high level of CAR (Fig. 7A), whereas OV-4 is mod-
est CAR expressor (Fig. 7C).
Gene transfer experiments utilizing SKOV3.ip1 and OV-4
showed that incorporation of recombinant RGD-containing
fiber protein into Ad5lucRGD virion dramatically improved
the ability of the virus to efficiently transduce these cells (Fig.
8A). At different MOIs tested, Ad5lucRGD-transduced cul-
tures of SKOV3.ip1 cells showed 30- to 60-fold increases in
luciferase activity over that of cells transduced with control
virus. Interestingly, while the fiber knob blocked more than 90%
of AdCMVLuc-mediated gene transfer, it could block only 15
to 20% of luciferase activity in Ad5lucRGD-treated cells.
The difference in transduction efficiencies demonstrated by
these two viral vectors was even greater, 300- to 600-fold, when
OV-4 cells were employed (Fig. 8B). As before, the fiber knob
used as an inhibitor of CAR-mediated cell entry did not have
a significant effect on Ad5lucRGD-mediated gene delivery,
strongly suggesting that this virus primarily utilizes RGD-inte-
grin interaction in order to bind to OV-4 cells.
We next evaluated the utility of the Ad5lucRGD vector in
the context of human ovarian cancer primary cells. In this
regard, recent human clinical trials have highlighted the dis-
parity between the efficacy of Ad vectors in various model
systems and in the clinical context, where rather low transduc-
tion efficiencies have been noted (1, 2). These findings suggest
the need to improve vector design as a general approach to
augment the therapeutic index of the cancer gene therapy
strategies. As integrins have been shown to be frequently over-
expressed by various epithelial tumors (for reviews, see refer-
ences 17 and 40), vector targeting to these cell surface recep-
tors can provide a means to achieve CAR-independent gene
transfer.
In our experiments, ovarian cancer cells obtained from two
patients were treated with both Ad5lucRGD and AdCMVLuc
in the presence or absence of blocking knob protein. The
results obtained corroborated our previous findings generated
with cultured cells. Note that luciferase readings in the lysates
of cells treated with AdCMVLuc were extremely low (Fig. 9A
and B), thereby indicating the inability of Ad vector containing
unmodified fibers to efficiently infect ovarian cancer cells.
Strong inhibition by the fiber knob on AdCMVLuc-mediated
luciferase expression suggests that the fiber-CAR interaction is
the only pathway this virus can use to infect this type of cell. In
marked contrast, Ad5lucRGD directed levels of transgene ex-
pression 2 to 3 orders of magnitude higher than those detected
FIG. 7. Flow cytometric analysis of human ovarian cancer cells. Expression
of CAR, a
v
b
3
, and a
v
b
5
integrins in SKOV3.ip1 or OV-4 cells was analyzed by
flow cytometry essentially as described in the legend for Fig. 3. Expression of
CAR in SKOV3.ip1 (A) and OV-4 cells (C) and of a
v
b
3
(thin line) and a
v
b
5
(heavy line) integrins in SKOV3.ip1 (B) and OV-4 (D) cells is shown. The results
for the negative control are shown by the dotted line.
FIG. 8. Comparison of the gene transfer efficiencies to cultured ovarian
cancer cells mediated by AdCMVLuc and Ad5lucRGD. Human ovarian cancer
cells SKOV3.ip1 (A) and OV-4 (B) were transduced with AdCMVLuc or
Ad5lucRGD at an MOI of 1 or 10 PFU/cell essentially as described for 293,
HUVEC, and RD cells. Recombinant Ad5 fiber knob protein was added to cells
prior to infection with the virus. Each datum point is the average of three
independent measurements obtained in one experiment.
FIG. 9. Transduction of primary cells isolated from ascitic fluid samples
obtained from ovarian cancer patients. Cells isolated from ascitic fluid samples
from two (A and B) ovarian cancer patients as described in Materials and
Methods were transduced with AdCMVLuc or Ad5lucRGD at an MOI of 1 or
10 in the presence or absence of blocking Ad5 fiber knob protein. The datum
points represent the means 6 standard deviations of three independent deter-
minations.
VOL. 72, 1998 GENETICALLY MODIFIED Ad5 VECTOR WITH EXPANDED TROPISM 9711

in AdCMVLuc-transduced cells. The knob blocked 20% of the
gene transfer at an MOI of 1 PFU/cell. No effect was observed
at an MOI of 10 PFU/cell. Thus, the ability to achieve signif-
icant enhancement of gene delivery via CAR-independent
pathway suggests the general utility of genetic retargeting of
Ad vectors for efficient tumor transduction.
DISCUSSION
In this report, we describe the generation and characteriza-
tion of recombinant Ad vector containing fibers with an
RGD-4C sequence genetically incorporated within the HI loop
of the carboxy-terminal knob domain. An effort to create such
a virus was undertaken in order to demonstrate the utility of
the HI loop of the fiber knob as an optimal site for incorpo-
ration of short peptide ligands, which would allow the virus to
bind to ligand-specific cellular receptors, thereby resulting in
altered or expanded tropism of the vector.
The interaction between cellular integrins and various pro-
teins containing an RGD tripeptide is one of the best charac-
terized interactions between macromolecules. This interaction
plays an important role in a variety of fundamental biological
processes, including cell adhesion and viral infection. In this
regard, it has been shown that the RGD motif contained in
adhesive proteins, such as fibrinectin, vitronectin, collagen, os-
teopontin, thrombospondin, fibrinogen, laminin, and von Wille-
brand factor (16, 29), allows efficient and specific interaction
between these proteins and integrin molecules. It is also known
that an RGD motif is present in some viral proteins including
the VP1 proteins of coxsackievirus (32–34) and foot-and-
mouth disease virus (13), the penton base protein of the ma-
jority of known Ad (25), the VP7 proteins of the African
horsesickness virus and bluetongue virus (7), the Tat protein of
human immunodeficiency virus (6), and the glycoprotein H of
herpes simplex virus (14). In some of these instances, this
tripeptide has been shown to play an important role in the
process of viral infection by mediating primary or secondary
interactions between the virion and cell surface-localized inte-
grins. Furthermore, recent studies showed that genetic incor-
poration of the RGD-containing sequences into chimeric hep-
atitis B cores (11, 36), poliovirus particles (30), bacteriophage
fd (19, 20), and human Ad virions (44) allows specific interac-
tion of these viral particles with cellular integrins, thereby
resulting in binding of aforementioned structures to cell sur-
face.
We utilized a similar genetic strategy in order to expand the
tropism of recombinant Ad vector with respect to cell types
which normally are refractory to Ad infection. Based on our
previous findings on accessibility of the HI loop-localized
FLAG peptide (21), we hypothesized that positioning of the
RGD-4C peptide in close proximity to the putative cell binding
domain localized within the knob of Ad5 fiber protein (45)
should make this ligand available for efficient interaction with
integrins on the cell membrane. By using an ELISA-based
binding assay, we have been able to show direct interaction
between the RGD motif of the fiber-RGD protein with puri-
fied integrin a
v
b
3
. This key finding provided a rationale for the
generation of recombinant Ad vector, Ad5lucRGD, contain-
ing such fiber-RGD proteins. The data generated with Ad5luc
RGD on several cell lines showed that this virus demonstrates
profiles of gene transfer significantly different from those by
the virus with unmodified fibers. This difference was especially
dramatic when CAR-negative cells were utilized for the gene
delivery experiments. Investigation of radiolabeled virus bind-
ing to the cells in vitro paralleled our gene transfer experi-
ments, thereby supporting the concept of augmented efficiency
of transgene expression as a result of more-efficient primary
interaction between the virus and the target cell.
In order to demonstrate the utility of the newly generated
viral vector for clinical applications in the context of gene
therapy, we employed Ad5lucRGD for gene delivery to cells
isolated from ascitic fluid samples obtained from ovarian can-
cer patients. Our experiments showed that in this model
Ad5lucRGD was able to direct levels of transgene expression
2 to 3 orders of magnitude higher that those mediated by
control virion containing unmodified fibers. These results
strongly suggest that recombinant Ad vectors containing fibers
with genetically incorporated RGD peptides may be of great
utility in the context of cancer gene therapy approaches based
on in vivo gene delivery. In addition, well-documented over-
expression of several types of integrins in tumor vasculature
(26) suggests that derivatives of Ad5lucRGD expressing ther-
apeutic genes may be utilized for eradication of tumors via
abrogation of their blood supply.
Successful utilization of the RGD tripeptide incorporated
into HI loop of Ad fiber protein for the purposes of vector
retargeting suggests that other peptide ligands may work just as
well in a context of the fiber molecule. In this regard, the
rapidly emerging technology of phage display libraries has
proved its utility as a means to identify peptides, which dem-
onstrate the ability to specifically bind to certain molecules on
a cell surface in vivo. This high-throughput method is based on
the capability of small peptide ligands to target a bacterio-
phage particle to previously characterized as well as to un-
known structures on a cell membrane. Recent successes in
phage biopanning in an in vivo context (3) strongly suggest that
this novel technology may provide an ideal source of targeting
peptides to be used for modification of endogenous tropism of
recombinant Ad vectors.
Whereas we have demonstrated the utility of small peptides
to be incorporated into the HI loop of the fiber knob, the size
restrictions of this locale have not been fully defined. In this
regard, the compatibility of the HI loop structure with protein
ligands of a larger size, such as, for example, single-chain
antibodies (scFv), would significantly expand the range of po-
tential targeting approaches. Furthermore, incorporation of
large polypeptide ligands into the HI loop, which connects
b-strands H and I involved in the formation of the cell-binding
site, may create a steric hindrance, thereby preventing direct
interaction of the fiber knob with CAR and resulting in elim-
ination of endogenous tropism of the virus. This, in turn, would
result in a new generation of truly retargeted Ad vectors,
capable of cell-specific gene delivery exclusively via CAR-in-
dependent mechanisms.
ACKNOWLEDGMENTS
This work was supported by the following grants: NIH grants RO1
CA-74242, CA-68245, and RO1 HL-50255; a grant from the American
Lung Association; and a grant from the U.S. Department of Defense,
DAMD 17-94-J4398.
We are grateful to Pierre Boulanger for making the recombinant
baculovirus AcNPV-PbWT available and Robert Gerard for providing
the plasmid pACCMVpLpA. We are indebted to Robert Finberg for
anti-CAR monoclonal antibody RmcB. We thank Paul Reynolds and
Alex Pereboev for fruitful discussions and proofreading of the manu-
script.
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